Biotests are methods where one uses living cells or organisms as tools to detect different analytes. Many of those methods utilize bacterial or yeast cells. Procaryotic organisms and especially Escherichia coli bacterium are very well characterized. Yeast cells are eucaryotic organisms and grow as single cells. The cultivation of yeast is easier than the cultivation of higher eucaryotes. Yeast cells grow in simple cultivation media and they do not need addition of complicated growth factors. The knowledge of yeast is expanding rapidly and comprehensive maps of genes are known. Hundreds of specific mutations for both bacteria and yeast are known. With knowledge of specific mutations it is possible to study the activity of specific reactions and metabolic pathways. For instance with antibiotic sensitive bacterial mutants trace amounts of antibiotics cause changes in the metabolism or in the membranes. Using antibiotic sensitive bacterial mutants, one is able to develop very sensitive tests to measure residual antibiotics from biological material. Bacteria and yeast with mutations in their DNA repair mechanisms, or mutants whose cell membranes might be porous for different small molecular weight substances, e.g., antibiotics, are more sensitive to genotoxic substances than wild type. Using different mutant strains, one is able to measure for the presence of antibiotics and toxic or mutagenic agents. Genetic engineering techniques can be used to transfer new characteristics into bacteria or yeast cells. The new characteristics can be provided by proteins which are encoded by viruses. The protein do not exist naturally in the target organism. Use of genetic engineering techniques expands the applicability of bacteria and yeast cells for use in biotests.
The universal genetic code of DNA is similar in each organism. The relationship between carcinogenicity and mutagenicity is the basis for using tests for mutagenic agents as prescreening tests for carcinogenic agents. Testing for carcinogenicity in animals is extremely expensive and time consuming. Use of tests for mutagenic agents as a quick screening method for carcinogenicity has raised hope and interest. The quick screening method for carcinogenicity would decrease animal-based carcinogenicity testing.
The AMES-test (Ames, B. N., McCann, J. and Yamasaki, E. (1975) Mutat. Res. 31, 347) is the test used most often to screen for mutagenic agents. The AMES-test utilizes Salmonella typhimurium as a test organism. Utilizing the AMES-test one is able to detect the genotoxicity of most mycotoxins, aromatic amines and polycyclic hydrocarbons. However, the Ames-test is not able to detect the genotoxicity of carcinogenic metal salts or chlorinated hydrocarbons. The S. typhimurium strains used in the AMES-test contain point mutations in the biosynthetic route of the amino acid histidine. As the bacteria are exposed to the action of mutagenic substance, a reversion mutation occurs in the gene for histidine biosynthesis and the bacterium starts to produce histidine endogenously. Endogenous production of histidine gives the cell the ability to grow on minimal growth medium containing no added histidine. A pitfall in the AMES-test is poor sensitivity and slow performance. In the AMES-test all other genotoxic changes such as those acting on enzymes remain undetected. The test is also rather expensive for each particular compound tested.
A test for the detection of genotoxic substances based on bioluminescence is known (Ulizur, S., Weiser, I. and Yannai, S. (1980) Mut. Res., 74, 113-121). In this method, dark mutants of Photobacterium leioanathi and P. fischeri are used. In the presence of genotoxic substances, these strains start to emit light. The theoretical background of the method remains somewhat obscure. It has been speculated that the effect of removing a repressor or preventing its formation combined with a change in the chromosomal DNA of the bacterium might trigger the formation of light producing proteins. Different genotoxic substances act with different rates in this test due to the variety of different classes of substances. This test is faster than the Ames-test but is by no means easier to use. The bacteria used in the test should produce light during long and varying periods of time (30 min to 10 h) depending on the substance. The bacteria used are not capable of stably emitting light, which makes the method somewhat problematic. Due to these facts, the method is not easily automated for use in routine work when there are a lot of specimens to be analyzed. Additionally, being of marine origin, the cultivation temperature of the bacteria is rather low, 15xc2x0 C.xe2x80x94it is not known how well the effect of genotoxic substances correlate to the effects on man whose body temperature is 37xc2x0 C.
Antibiotics, used as medicines against microbial invasion, are detected from body fluids in order to study the dosage and penetration of the medicine. The effective therapeutic range of the antibiotic is often rather narrow and the risks due to overdosage might be large. It is also important to measure the presence/concentration of antibiotics in meat and cow milk due to symptoms in people with allergies to antibiotics. The cow milk used in cheese production should not contain antibiotics due to the fact that cheese making bacteria are not able to grow in antibiotic-contaminated milk. Common methods for detecting antimicrobial medicines are microbiological methods performed on agar. A direct method is to measure the inhibition of the growth of sensitive bacterial strains. One can also measure some metabolic parameters, such as acid production of a sensitive strain of bacteria, using proper color indicators.
Typical examples of agar diffusion tests are cylinder, hole or disk methods. The difference between these tests is in the way the sample is applied to the agar and also in the way the bacteria are utilized in the test.
Since microbiological methods utilize bacteria or their spores, the sensitivity of the test bacteria is of utmost importance. In the tests described above compromises had to be made in the choice of a suitable test strain since great sensitivity against antimicrobial agents and other characteristics needed for the test strain have not been found in the same strain of bacteria.
Major drawbacks when using microbes in antibiotic residue tests are slow speed and insensitive performance. In these methods one controls the growth of a test strain and thus the test cannot be performed in an hour. This is due to the fact that growth of microbes is a slow process even in its fastest mode. In addition, in many cases spores or freeze-dried microbes are used which make the tests even slower to perform.
Antibiotic detection methods based on bioluminescence measurement are known. Ulizur (1986, Methods Enzymol., 133, 275-284) describes three different ways to use bioluminescence for the detection of antimicrobial agents: a) lysis-test, b) induction test and c) bacteriophage test. In the first one, the lux-genes isolated from Vibrio fischeri produce luciferase protein which in the presence of substrates produces light. The genes have been cloned into a plasmid and transferred to Bacillus subtilis. The B. subtilis strain utilized is sensitive to antibiotics which affect bacterial membranes. Examples of such antibiotics are penicillins and cephalosporins. In the lysis-test, the lux-gene-containing B. subtilis is grown together with a test sample. If the test sample contains an antibiotic, the synthesis of cell wall components is prevented and the bacteria are lysed. Thus the culture yields lower light emission when compared to a culture lacking the test sample.
The induction test utilizes dim mutants of P. phosphoreum bacteria, which do not produce light. The induction test and other bioluminescence tests developed by Ulizur are based on exploitation of the chromosomal DNA of the target cell. Antibiotics affecting protein synthesis are detected in the induction test. When the bacteria are incubated together with compounds that bind to DNA, the bacteria start to produce light, i.e., protein synthesis is initiated. If there is any antibiotic present affecting the protein synthesis then there is a decline in light emission. The amount of antibiotic present is quantitated when compared to a blank without antibiotic. The induction test will not detect antibiotics that affect DNA synthesis and its basis is obscure. To perform the induction test it is essential to add minimal salts such as Ca2+ and Mg2+ ions which are known to diminish or completely prevent the action of aminoglycosides (streptomycin, kanamycin, neomycin, erythromycin). In addition, the induction parameters are very strict. If samples contain other antibiotics (for instance nalidixic acid) or other substances triggering light production there may be problems in the interpretation of the results. The amount of bacteria in the test is a critical parameter. If the concentration of bacteria in the test is too high, the culture has to be aerated due to the absolute requirement of oxygen for the bioluminescence reaction in these bacteria. Additional problems include a great number of potential inducers, special measuring devices, and reproducibility of results.
The bacteriophage test can be utilized to detect antibiotics affecting DNA synthesis, transcription and translation. In this test, wild-type, light-emitting P. phosphoreum bacteria are infected with lytic bacteriophages. In the presence of an antibiotic, new infectious phages cannot be synthesized due to the fact that DNA-, RNA- or protein synthesis is blocked. In the presence of an antibiotic, the light emission is unchanged compared to the initial light level. However, if no antibiotic is present, the phages rapidly multiply and inactivate the host bacteria thus making it incapable of producing light. The bacteriophage test is difficult to perform since it is necessary to add phages (sometimes with different titers) to the assay mixture and the addition of antibiotic has to be carefully timed. The bacteriophage test suffers some of the same problems discussed with the induction test. Notably, the composition of assay mixture and the amount of bacteria used in the assay.
The methods discussed above can be used to reveal the presence of antibiotics generally but not to reveal individual antibiotics. By changing measuring conditions or by adding enzymes to degrade certain compounds, one is able to block the effect of some antibiotics. There is a great demand for fast and simple methods to detect heavy metals, toxins, or food additives. At the moment detection of these compounds must be performed in central laboratories. The devices for detecting heavy metals, toxins, or food additives are extremely expensive and need specially trained personnel to use them. Quick, qualitative tests performed in the field could screen for those samples which need more sophisticated instrumentation and research. Thus, the pressure on central laboratories would diminish and determination of problematic samples would be faster.
A commercial xe2x80x9cMicrotoxxe2x80x9d test is able to detect toxic substances from environmental specimens. This test is based on the use of light emitting P. phosphoreum bacteria. A sample to be analyzed is incubated together with bacteria and the presence of a toxic substance is evaluated from a decreased level of light produced by the bacteria when compared to controls. A severe drawback with this test is the need for high salinity (2%) by the organism. High salinity has been shown to decrease the biological effect of heavy metals. In addition, an incubation temperature of 15xc2x0 C. can be an obstacle.
In addition to the Microtox test, several tests utilizing whole animals or animal cell lines have been developed to measure toxic substances. Pitfalls in these methods include complicated cultivation of cells, slow performance and a need for skilled personnel.
A test should be able to detect toxic and mutagenic substances from different waters such as from waste-, consuming-, raw- and groundwater and from water for refreshment purposes. In addition, water needed for industrial processes, food processes as well as raw water needed for pharmaceutical industry are of interest. The test should be able to evaluate samples from ground sediments and air for their toxicity and mutagenicity. The raw material used in food industry as well as quality control of food stuffs needs attention. From certain waters one should be able to detect the organic material which could be used for respiration and biosynthesis purposes by microbes contaminating the water. The organic material can be simple sugars, organic acids, peptides or proteins, compounds containing amino or phosphate groups linked to carbon chain etc. There is a need for rapid, non-expensive tests for these kind of compounds since the conventional methods take several days to complete in order to be able to evaluate the quality of water used for various purposes.
The invention described here is based on known and accepted principles of gene expression, the factors affecting gene regulation, and on the use of recombinant-DNA in organisms such as bacteria and yeast.
Gene technology has made it possible to use bacteria and yeast cells as hosts to produce proteins that these organisms do not produce naturally. Several kinds of recombinant-DNA vectors have been prepared for this purpose. Usually, the recombinant-DNA vectors are extrachromosomal plasmids. Recombinant-DNA plasmids can contain several genes and they can replicate independently of host chromosomal DNA. Recombinant-DNA techniques are utilized to transfer foreign genes into new host cells. The gene of interest can be cloned into a plasmid vector with restriction enzymes and DNA ligase. Following cloning, the recombinant-DNA plasmid is transformed into a bacterial host using methods well known in the art. For expression in a eukaryotic cell, the recombinant-DNA plasmid usually contains DNA from bacteria, viruses, and eukaryotes. Recombinant-DNA is transferred into eukaryotic cells using techniques well known in the art, such as calcium-phosphate precipitation or electroporation.
Many recombinant-DNA plasmids have been developed during the last few years where a gene of interest has been placed under control of a strong promoter. A goal has been to create high expression of a foreign gene in an organism such as E. coli. In an E. coli bacterium, the production of protein from the gene of interest can account for as much as 25% of the total cellular protein (Caulcott and Rhodes, 1986, Trends in Biotech., June, 142-146). Overexpression of a foreign gene can be deleterious to the host cell or its metabolism. To overcome the potential problem of overexpression, plasmids with regulatable promoters were developed, and the gene of interest is placed under the control of the regulatable promoter. The production of protein from the gene of interest can be turned on when the microbes achieve an optimal growth phase. Cultivation of the microbes to the optimal growth phase is performed under non-stressing conditions. Expression of the foreign gene is usually controlled by adding a chemical to the medium which activates the regulatable promoter. For example, plasmid pCSS108 (Korpella and Karp, Biotechnol Lett., 10(6), 1988, 383-388), shown in FIG. 6, contains a bacterial luciferase gene under the control of a regulatable promoter. Expression of bacterial luciferase is induced by isopropyl-xcex2-D-thiogalactopyranoside (IPTG) added to the culture medium. In addition, physical parameters, such as an increase or decrease in incubation temperature might regulate protein production.
The plasmids commonly used contain one or more resistance determinants, with which to select from a large population of cells only those which contain the plasmid. The resistance determinants helps the cell to survive in circumstances which are poisonous to other cells. A selection agent is added to the growth medium and cells which contain the plasmid are able to grow, but cells which lack the plasmid are not able to grow. The resistance determinant is a gene which encodes a protein that degrades or otherwise inactivates the poisonous factor. Examples of such poisonous factors are antibiotics which are present in the growth medium. Several genes encoding resistance factors are known. The resistance factor used most often is the gene encoding xcex2-lactamase. xcex2-lactamase degrades penicillins or xcex2-lactams which are penicillin derivatives. As a result of xcex2-lactamase enzyme activity, the poisonous character of penicillin is lost and bacteria can grow. Other commonly used resistance genes include those encoding chloramphenicol acetyltransferase, kanamycin acetyltransferase and tetrahydrofolate reductase.
Depending on the type of cells chosen, genes which carry the ability for the cell to grow in the presence of tetracyclin, erythromycin, spectinomycin, streptomycin, sulfonamides, neomycin, thiostrepton, viomycin and colicins can also be used. Some resistance factors that eliminate or change the heavy metal present in the medium can also be used. Selection pressure in favor of a cell containing a plasmid can also be achieved by transferring a gene encoding a function which complements a growth defect in the organism. Normally, such growth defects arise from a defective gene in the chromosome of the organism. These genes are normally those which encode for proteins participating in an amino acid biosynthesis pathway. An essential amino acid is removed from the growth medium and the cell cannot grow unless the amino acid biosynthesis gene is present. The gene will be present in the cell if the plasmid is present. When the gene is expressed the amino acid biosynthesis pathway will be complemented and the cell can grow. The plasmids can also encode genes of other vital functions in the cell. An example of such genes include genes encoding proteins that participate in the formation of the cell wall.
The copy number of various plasmids inside the cell can vary from one to several hundred, to over a thousand. A plasmid often used, pBR322, has a copy number of about 60 whereas a derivative of it, pUC8, has a copy number of about 500. A reason for the large difference between two related plasmids is due to a one base pair mutation in the origin of replication (ori) sequence of the plasmid (Chambers et al., 1988, GENE, 68, 139-149). The copy number of a plasmid can be artificially increased at a suitable phase of growth by constructing a vector where ori is placed under the control of a strong and regulatable promoter. At present several plasmids are known whose copy number can be artificially shifted up during the growth of microbes. These plasmids are mainly used in industrial processes to produce foreign recombinant proteins in large quantities. Thus the use of these run-away replication vectors for purposes described above does not rule out the possibility of using them in this invention for measuring different agents which affect the cell. As examples in this invention we describe different run-away plasmids with which a change in copy number is possible. Run-away plasmids studied and used to produce foreign proteins belong to series pOU. The origin of replication region of pOU plasmids has been put under the control of strong and regulatable PR promoter of phage lambda (Larsen et al., 1984, GENE, 28, 45-54). The PR promoter of phage lambda is regulated by a repressor protein, cI857, which is destroyed by heating to 42xc2x0 C. The protein can be produced from a lysogenic phage, i.e., a phage which is integrated in the chromosome of the host cell, from a plasmid where the coding sequence has been introduced or from another plasmid which belongs to a different incompatibility group. Plasmids of different incompatibility groups of are plasmids which are able to replicate independently without the presence of another plasmid in the same cell. When the repressor protein has been destroyed, PR promoter is turned on and without control it starts to produce proteins called copB and repA (originating from low-copy number plasmid R1) as well as transcription products of these and the copA gene. These factors and especially the overproduction of repA protein result in enhanced or even uncontrolled production of the plasmid DNA in E. coli bacterium.
Yeast as well as bacteria are single cell organisms but yeast differ from bacteria by being eucaryotic cells. Compared to higher eucaryotic cells yeast are far better characterized from the genetic point of view. The genetic maps of Saccharomyces cerevisiae and Schizosaccharomyces bombei are already known in great detail (Petes, 1980, Ann. Rev. Biochem., 49, 845-876). In addition, powerful methods to transfer genes into yeast are known. Thus, yeast are commonly used hosts for rec-DNA.
Four types of rec-DNA vectors are used with yeast: integration plasmids (YIp), episomal vectors (YEp), replicating vectors (YRp), and artificial chromosomes. The integrating vectors of yeast can contain DNA originating from bacteria and part(s) of yeast genes. This type of plasmid integrates exactly at certain point(s) in the yeast chromosome. The replicating yeast plasmids contain DNA from bacteria, part of yeast DNA and a specific area from yeast chromosome. The specific area from the yeast chromosome is responsible for the replication of the plasmid.
The specific area from the yeast chromosome permits the plasmid to replicate as extrachromosomal DNA molecule in the yeast cell. The episomal plasmids contain DNA from bacteria, a yeast gene and a part or the whole 2 micron plasmid of yeast (Hollenberg, 1982, Current Topics in Microbiology and Immunology, 96, 119-144). Artificial chromosomes are linear DNA vectors which are not well suited for expression of heterologous proteins.
A plasmid for yeast whose copy number can be regulated has been described. Centromeres are needed in yeast during the partition of a chromosome in mitosis and meiosis phases. Centromeric DNA (CEN3) has been extracted and transferred under the control of alcohol dehydrogenase promoter (ADH2). The ADH2 promoter is repressed by glucose. The action of a CEN3 plasmid can be controlled by the carbon source used to cultivate yeast. When glucose is used as carbon source the ADH2 promoter is repressed and CEN3 works normally by balancing the plasmid structure (YRp) during mitosis. If the carbon source in the growth medium is changed the plasmid starts to replicate and the copy number can increase up to one hundred per yeast cell (Chlebowicz-Sledziewska, E. and Sledziewska, A., 1985, GENE, 39, 25-31).
The expression vectors used in yeasts normally contain the following strong regulatable promoters: alcohol dehydrogenase isoenzyme I (ADHI) gene promoter, phosphoglycerol kinase (PGK) promoter, repressible acid phosphatase (PHO5) promoter and the promoter for xcex1-factor. ADHI is a yeast cytoplasmic enzyme. ADHI produces ethanol from acetaldehyde and needs NADH as a cofactor. When yeast cells are cultivated in the presence of glucose there is at least 1% ADHI protein from the total amount of proteins in yeast. The PGK promoter can be controlled by the carbon source (for example glucose) used. The expression of PHO5 can be prevented by the addition of inorganic phosphate and activated by eliminating inorganic phosphate from growth medium. The control of PHO5 happens through a special regulation apparatus, which is formed from PHO2, PHO4, PHO80 and PHO85 gene products (Bosfiana, K. A., 1980, Proc. Natl. Acad. Sci. USA, 77, 6541-6545). Some mutants (PHO4 and PHO80) are known, which can be activated by a simple change in temperature. These mutant yeast cells grow at 35xc2x0 C. and do not produce acid phosphatase enzyme even if inorganic phosphate is absent in the medium. If the cultivation temperature is shifted down, acidic phosphatase is produced efficiently whether there is phosphate or not in the medium. This system to control the protein production by a change in the cultivation temperature has been used to produce, for instance, interferons (Kramer et al., 1984, Proc. Natl. Acad. Sci USA, 81, 367-370).
The use of higher eucaryotes as host cells for rec-DNA vectors to produce foreign proteins is rapidly expanding. The goal of such use is to produce proteins of eucaryotic origin in large quantities. In an optimal expression system it would be possible to produce proteins in several different types of cell lines. A fully regulatable expression system for protein production would be an ideal solution. The regulatable promoters used most often work only in certain host cell systems. The regulation of these promoters is poor and the expression vectors are based on DNA of tumor producing viruses, thus there also exists certain risks in their uses.
In higher eucaryotes gene expression can be regulated with the help of the following: simian virus (SV40) T-antigen, metallothionein genes, heat-shock genes, glucocorticoid hormones, DNA methylation or with anti-sense RNA. The antigen produced by SV40 controls its own transcription. T-antigen is produced in large amounts immediately after the virus has infected the target cell. Later the T-antigen binds to its own promoter and prevents transcription. If SV40-vectors are used for cloning, regulation of the T-antigen can be prevented by using a suitable temperature sensitive T-antigen mutant. In these cases T-antigen mutants produce T-antigen normally at high temperatures but the production is prevented at room temperature (Rio et al., 1985, Science, 227, 23-28).
Metallothioneins are proteins which bind heavy metals. Many eucaryotic cells produce these proteins in the presence of heavy metals. It has been estimated that there is an increase over fifty fold in the production of metallothioneins when cadmium is added to the growth medium to a concentration of 4xc3x9710xe2x88x926 molar (Hamer, D. H. and walling, M. J., 1982, J. Mol. Appl. Genet., 1, 273-288). The protein production induced by cadmium can be further increased by using low Cd2+-content growth media.
Many promoters of heat-shock genes have been shown to be applicable and well regulated in several different cell lines. The regulation of these promoters is performed simply by shifting the growth temperature. The genes are activated at high temperatures and produce proteins. At low temperatures the proteins are produced in low amounts or not at all. The best studied example is the heat-shock system of common fruit-fly, Drosophila melanogaster, in which a rise in temperature from 25xc2x0 C. to 37xc2x0 C. causes the cessation of normal protein production and the heat-shock proteins start to emerge. A major heat-shock protein is hsp70. The regulation mechanisms of heat-shock protein expression are not well known. By using heat-shock promoters (hsp70), it has been possible to increase the production of hGH (human growth hormone) up to 1200-fold compared to unactivated cells (Dreano et al., 1985, GENE, 49, 1-8).
The invention described here uses procaryotic and eucaryotic organisms, which have been carefully selected and which contain applicable rec-DNA vector constructions. By turning on the synthesis of DNA, RNA or proteins under strict control one is able to measure or detect either directly or indirectly all those factors which affect on the synthesis machineries described above. As the basis of measurement one can use the protein product encoded by the rec-DNA vector, the marker protein or its activity or the overall metabolic activity. By activating the replication of rec-DNA vector in a controlled fashion one is able to measure the amount of DNA formed directly by using radioactive labels or with flow-cytometric techniques.
One is able to prepare suitable rec-DNA vectors for the measurement of different classes of chemicals depending on the target of the chemical. It is possible with the aid of E. coli bacteria containing runaway-replication type plasmids to quantitate, for instance, compounds inhibiting DNA synthesis (nucleotides) and DNA replication as well as those compounds binding to DNA like several cancer drugs. Replication of runaway-replication plasmids is controlled, for instance, by an inducible promoter, PRE, of phage lambda. Thus, DNA synthesis and the replication of a plasmid can be triggered at a predetermined point of time. The analytes to be measured can be linked directly to this regulatable and strong DNA biosynthesis which is not dependent on cell division. If the synthesis or replication of DNA is inhibited the result is seen in the copy number of plasmid. The copy number may be the same or even decreased compared to the initial stage. In the uninhibited control cells the copy number of plasmid per cell may increase rapidly. The change in copy number can be measured either directly by measuring the amount of DNA or indirectly by measuring the amount of gene products or the activity encoded by the plasmid DNA. It is possible to determine agents that have very different mode of action on the cell with the aid of this kind of a plasmid. This is due to the fact that one can also engineer a gene encoding a marker protein under the control of a strong and regulatable promoter, the expression of which is measured in the test. Thus everything affecting DNA, RNA, proteins, or their biosynthesis can be measured. If one wants to develop a broad range of tests which covers agents affecting cell wall, nucleic acids, proteins and metabolism an ideal means of detecting these agents is based on this kind of a runaway-replication plasmid. In these cases cells are allowed to replicate after which the promoter regulating the replication is activated. Simultaneously, or after a certain period, the promoter regulating a gene encoding a marker protein is activated. A vector with similar characteristics can be developed for eucaryotic cells.
Another kind of approach is to use plasmids whose replication is tied to host cell division. Rec-DNA multicopy plasmids in which the gene encoding the marker protein is under the control of a strong and regulatable promoter can be used to detect agents that affect cell membranes, proteins and metabolism. Agents affecting DNA or cell membranes can be detected with the system if actively dividing cells are used. The multicopy plasmid is synthesized for daughter cells and the system is sensitive to agents affecting DNA. Actively dividing cells are also sensitive to agents affecting the cell membranes. If the strong promoter regulating the expression of the marker protein is activated the system ill then also be sensitive to agents affecting the mRNA and protein synthesis.
As a special application, when genes encoding luciferase are used, one can determine agents that affect energy metabolism. This is due to the fact that the reactions catalyzed by luciferases use energy-rich substances of cells. Agents that can affect the energetic state of the cell on all biosynthetic levels (replication, transcription and translation) or in metabolism, can be determined with the aid of bioluminescence, i.e., formation of light emission by the cells. A special case is bacterial luciferase, which uses central products of metabolism, NAD(P) and FMNH2. Another special case is fire-fly and click beetle luciferases, which use a central metabolite, ATP, for light production.
The nature of the invention described here makes it possible to use very different kinds of measuring modes for example spectrophotometric, fluorometric, luminometric and visual methods. Spectrophotometric methods can be an alternative when there is a gene cloned into plasmid whose product can be measured by monitoring the change in color such as xcex2-galactosidase, alkaline phosphatase, amylases, peroxidases, glucuronidases or oxidoreductases. Fluorometric methods utilize fluorescent substrates developed for various enzymes, thus yielding somewhat greater sensitivity compared to spectrophotometric techniques. The luminometric method is performed with the aid of genes encoding either bacterial or beetle luciferases. Several luminescent bacterial species exist such as V. harveyi, V. fischeri, P. leiognathi, P. phosphoreum, Xenorhabdus luminescens etc. Examples of luminescent beetles are Luciola minarelica, Photinus pyralis, Pyrophorus plaaiothalamus etc. There several eucaryotic species in the sea which luminesce, such as marine ostracod Vargula hilgendorfii, jellyfish Aeguorea victoria, batrachoidid fish Porichtys notatus, pempherid fish Parapriacanthus ransonneti etc., which could be useful in the future for various applications. Here an advantage over spectrophotometric and fluorometric measurement is the extremely sensitive detection of light emission. An important benefit in luminescent methods is the possibility to calibrate internally the measurements by using other intracellular genes which encode luciferase emitting a different color which could be measured with a special two wavelength-detecting apparatus. The other gene can be cloned in the same rec-DNA vector, in another vector belonging to a different incompatibility group, inserted in the host chromosome, or in a phage etc. An example is the click beetle luciferases, which emit four different colors. The wavelengths range from 547 nm to 593 nm (Wood et al., 1989, Science, 244, 700-702). The other gene resulting in different wavelength can be put under an inducible production system (indicator xe2x80x9cgenexe2x80x9d) or it can be expressed constitutively (internal standard) to compensate possible secondary effects arising from heterologous samples. The use of a simple color indicator is useful in cases where there is no need for high sensitivity but where the simplicity and fast performance are more important. If the changes in cell metabolism are to be detected one can use for example tetrazolium salts which form a low-solubility formazan color when reduced. In these cases genes encoding dehydrogenases or oxidoreductases act as mediators of reducing quantities to yield the intense color of formazan. Also immunological methods (antibodies) coupled to sensitive measuring systems (RIA, FIA) are possible. Use of radioactive labels and flow cytometry in detecting the end point of the test are possible.